Extraction and Electrophoresis of Histones

Chromosomes

Morphotype	Activated chromatin Euchromatin	Nonactivated chromatin, Facultative and obligate heterochromatin, euchromatin
Structural organization	Less condensed, unfolding of functional domains (2040 kbp) exhibit	Highly condensed
DNA methylation (CG sites)	DNase-I-sensitive sites mv-sites unmethylated	mv-sites methylated
Nucleosomes	DNase-I-sensitive sites	DNase I resistant
Histones	H1-deprivated; core histones highly acetylated	H1-enriched; association with special H1 isofores, e.g., H5; H2A/H2B underacetylated; eventually H2A modified by ubiquitin
HMG 14/17	Present	Absent
HMG 1/2	Present	Absent
Transcription RNAP/RNP	Presence of RNAP and RNP depends on the actual transcription state	No
TABLE 1. Properties of chromatin

Materials

• Saline citrate (1/10 SSC)
• 1.0 N H₂SO₄
• Refrigerated preparative centrifuge
• Absolute ethanol
• β-mercaptoethanol
• 0.01 M sodium phosphate buffer, pH 7.0 + 1% (w/v) SDS + 0.1% (v/v)
• β-mercaptoethanol
• 10% acrylamide gels (10%T:5%C) with 0.1% (w/v) SDS
• 7% (w/v) acetic acid
• 0.25% Coomassie Blue

Procedure
Dissolve crude chromatin in cold dilute saline citrate (0.015 M NaCl + 0.001 M sodium citrate) to a final DNA concentration of 500 µg/mL.

1. Stir the solution on ice and slowly add ¼ volume of cold 1.0 N H2SO4. Continue stirring for 30 minutes.
2. Centrifuge the suspension at 12000 xg for 20 minutes at 4°C. Save the supernatant. For maximum yield, break up the pellet, resuspend in fresh, cold 0.4 N H₂SO₄, re-extract, centrifuge, and add the resulting supernatant to the first.
3. Add 4 volumes of cold absolute ethanol to the supernatant and store for 24 hours at –10° C to precipitate the histone-sulfates.
4. Collect the precipitate by centrifugation at 2000 xg for 30 minutes.
5. Decant as much of the alcohol as possible, and resuspend the pellet in cold absolute ethanol.
6. Centrifuge at 10000 xg for 15 minutes.
7. Collect the pellet and freeze dry for later analysis. To continue with the electrophoresis, carefully weigh the histone protein sample and dissolve in 0.01 M sodium phosphate buffer with a pH 7.0 and containing 1% sodium dodecyl sulfate and 0.1% β-mercaptoethanol; the final volume should contain approximately 300 mg of protein in 100 mL of buffer.
8. Prepare the electrophoresis chamber with a 10% acrylamide gel with 0.1% SDS.
9. Add separately 25 µL of the dissolved protein and 25 µL of protein standards to:
   50 µL of 0.1% SDS, 0.1% β-mercaptoethanol in buffer
   5 µL of β-mercaptoethanol
   1 µL of 0.1% bromophenol Blue in water
10. Mix thoroughly and apply the histone extract and protein standards to separate wells of the electrophoresis gel.
11. Separate the proteins in the anode direction (anionic system).
    • The addition of SDS anions to the proteins results in negatively charged proteins, which will separate according to molecular weight.
    • Electrophoresis is carried out in the standard manner. The buffer utilized is Laemmli.
    • 0.025 M Tris-0.192 glycine and 0.1% SDS, pH 8.3.
    • Proteins are separated by a current of 3–4 mA per gel until the bromophenol marker reaches the bottom of the tube (about 7 hours at 3 mA, and 4 hours at 4 mA).
12. Stain the gels with 0.25% Coomassie Blue for 2 hours.
13. Destain and store in 7% acetic acid.
14. Scan the gels and determine the molecular weights of each component.

Notes
Preparation of a total histone fraction from nuclei is normally accomplished by extraction with a dilute acid or a high-molarity salt solution. The acidic extraction removes histones from DNA and nonhistones immediately, while the dissociation of chromatin in salt solutions will require further purification. In either event, the histones themselves are subdivided into 5 major types, designated as H1, H2, H3, H4, and H5. H2 dissociates into 2 peptides, which are thus designated as H2A and H2B. The classification of histones is based on their electrophoretic mobility.

Nonhistone proteins can also be extracted and separated by electrophoresis. Whereas histones have only 5 major types, nonhistones are extremely heterogeneous and up to 500 different proteins have been identified from one cell type, while the major proteins comprise less than 20 types. The extraction of chromatin DNA was possible with the 7 M urea-3 M NaCl extraction. Further analysis of DNA will be undertaken as part of a later lab exercise (on transcription), and the DNA sample from this lab may be kept lyophilized and frozen until that time.

For our current needs, it is sufficient to note that the genes are composed of DNA, and that various specific regions of the DNA/genetic information can be physically isolated to a specific locus on a chromosome. This, in turn, is readily observed and correlated with banding patterns, such as those in the fruit fly polytene chromosomes.